The Aim Of Biosafety Training Is To Increase Your Ability To Recognize And Reduce Hazards In a BSL1 Lab Think before you do anything What could happen? What is the worst thing that could happen? What can I do to prevent it? What will I do if I can t prevent it?
What is Biosafety? Biosafety describes the prac>ces and containment under which poten>ally biohazardous materials can be used safely. What are poten>ally biohazardous materials? InfecBous agents or pathogens such as bacteria, viruses, fungi & protozoa Recombinant DNA (rdna) such as genes cloned into plasmid or viral vectors Do the genes encode toxins, an>bio>c resistance or oncogenes? Will they be expressed or used to generate transgenic organisms? Toxins such as tetrodotoxin, ricin and botulism toxin 2
What is Biosafety? The goal of a Biosafety program is to protect individuals who work with poten>ally biohazardous agents from exposure to those agents. to ensure the safety of others in the work area and larger community To ensure that biohazardous materials are not released into the environment Biosafety defines a code of safe working prac>ces and lab design to meet these goals. 3
What is a Biological Safety Level? A Biosafety Level can be assigned to lab work involving poten>ally biohazardous materials Each Biosafety Level describes the prac>ces and containment that will reduce the risk of exposure to poten>al biohazards. There are 4 levels of biosafety Level 1 represents the prac>ces and containment required for biohazards that pose the lowest hazard. Level 4 is reserved for labs using materials that pose the greatest hazard. High Hazard BSL 4 BSL 3 BSL2 Low Hazard BSL1
How is a Biosafety Level DesignaBon Determined? A risk assessment is used to 1. Consider What poten>ally biohazardous materials will be used How will these materials be manipulated? AND 2. Determine Primary barriers and safety equipment to be used Secondary barriers or lab design that is required High Hazard BSL 4 BSL3 BSL2 Low Hazard BSL1
Resources for Risk Assessment and DeterminaBon of Biological Safety Level In the U.S., biosafety levels (BSL) are defined in two documents: 1. Biosafety in Microbiological and Biomedical Research Laboratories (the BMBL) from the Centers for Disease Control and Preven>on (CDC). 2. The NIH Guidelines for Research Involving Recombinant or Synthe>c Nucleic Acid Molecules (the NIH Guidelines) from the Na>onal Ins>tutes of Health (NIH). BSL 4 BSL 3 BSL2 BSL1
So What is a Biosafety Level 1 Lab? Let s look at examples of What poten>ally biohazardous materials will be used How will these materials be manipulated? AND Determine Primary barriers and safety equipment to be used Secondary barriers or lab design that is required High Hazard BSL 4 BSL3 BSL2 Low Hazard BSL1
What is a BSL1 Lab? CharacterisBcs of the biohazardous material BSL1 is appropriate when agents that are generally not pathogenic are in use: E. coli K12 Saccharomyces cerevisiae (yeast) plasmid cloning vectors Biosafety Element Biosafety Level 1 CharacterisBcs of the biohazardous material Planned manipula>ons Not known to consistently cause disease in healthy adults* Standard Microbiological Prac>ces * many agents not ordinarily associated with disease may cause infec>on in the young, the aged and immunocompromised individuals. Primary containment and protec>on Facili>es design (secondary containment) PPE: gloves, lab coat and eye protec>on are used Handwashing sink, emergency shower and eyewash, autoclave
What is a BSL1 Lab? What is going to be done with these materials? Standard Microbiological Prac>ces should be observed. Biosafety Element Biosafety Level 1 Characteris>cs of the biohazardous material Not known to consistently cause disease in healthy adults The prac>ces followed at higher biosafety levels are based on these standards. Planned manipulabons Standard Microbiological Prac>ces What are Standard Microbiological PracBces? Primary containment and protec>on PPE: gloves, lab coat and eye protec>on are used Facili>es design (secondary containment) Handwashing sink, emergency shower and eyewash, autoclave 9
Standard Microbiological Prac>ces Are prudent pracbces that should be pracbced every day in every laboratory safety program We will discuss some of these in the next few slides Wash hands a_er handling biologicals, taking off gloves and before leaving the lab. No ea>ng, drinking, smoking, or applying cosme>cs in the lab. Wear clothing (sleeves, scarves, shoes, jewelry) appropriate to your tasks. Tie hair back. Always use mechanical pipebng devices (never mouth pipece). Use personal protec>ve equipment (lab coats, safety glasses and gloves). Decontaminate work surfaces daily and a_er spills. Use cau>on when handling sharps, such as broken glass and razor blades Use procedures that minimize the forma>on of aerosols and splashes. Place all solid biological waste in red autoclavable bags for disposal. Liquids must be disinfected before sink disposal. Be familiar with instruc>ons on spill clean up before a spill happens! 10
Standard Microbiological PracBces Wash hands a_er handling biologicals, taking off gloves and before leaving the lab. No ea>ng, drinking, smoking, or applying cosme>cs in the lab. Wear clothing (sleeves, scarves, shoes, jewelry) appropriate to your tasks. Tie hair back. Always use mechanical pipebng devices (never mouth pipece). X 11
Decontaminate Work Surfaces Work surfaces must be decontaminated. Before you begin to work every day A_er a spill, splash or any contamina>on A_er you are finished with your work or at the end of the day Use Simple Green, Lysol or 10% bleach When you apply the disinfectant, be careful not to generate splashes Allow the disinfectant to remain on the bench for at least 5 minutes before you wipe it up Dispose of the paper towel in the biohazard waste bag Wash your hands
Biohazardous Waste Management PracBces All solid biohazardous waste must be placed in red biohazard bags Do not overfill! Empty when 2/3 full These will be incinerated or steam sterilized before disposal. All liquid biohazardous waste must be sterilized by autoclaving or treatment with approved disinfectant 10% bleach for >20 minutes- before disposal.
Needles and Sharps PrecauBons Because percutaneous exposure (through the skin) is a route of transmission of many pathogens, extreme cau>on should be taken with contaminated needles and other sharps. Sharps include items such as razor blades and scalpel blades broken glass* plas>c pipeces and >ps microscope slides
Needles and Sharps PrecauBons Wear gloves *Broken Glass Do NOT acempt to pick up pieces of broken glass with your gloved hands. The risk of being cut is too great. Use tongs or a brush and dust pan- or even 2 pieces of s>ff cardboard- to collect broken glassware.
Proper Disposal of Needles, Sharps and Broken Glass Contaminated sharps disposal Do NOT overfill Hard walled plas>c containers labeled with the biohazard symbol Waxed cardboard take out containers Large pieces of broken glass or sharps must be placed in a puncture resistant container (such as a cardboard box) lined with a biohazard bag, clearly marked "GLASS AND SHARPS.
Spill Clean Up Procedures Use appropriate PPE: gloves, goggles and lab coat If there is broken glass, it should be cleaned up using tongs or a dust pan and placed into either a sharps container or a biohazard bag inside a box.
Spill Clean Up Procedures, cont d Spills should be treated with disinfectant (10% bleach) for >20 minutes. Use absorbent material soaked in disinfectant to cover the spill Cover the spill with paper towels and then pour on disinfectant Be careful not to generate splashes and aerosols! The paper towels should be placed in the biohazard waste. Repeat the disinfec>on process.
Spill Clean Up Procedures, con t All PPE should be placed into biohazard waste and hands washed thoroughly. Report the incident to your instructor immediately. Complete an Incident and Accident Report Form These are in the back pocket of the Bisafety Manual and at fandm.edu/biosafety
What To Do In The Event Of An Exposure Immediate First aid Wash the affected area with disinfectant for >15 minutes. For membrane exposure (eyes, nose, mouth) flush with water for >15 minutes. Cover the wound. Exposure may require an immediate evalua>on by a medical prac>>oner. Go to Appel Health or Lancaster General Hospital
What to do in the event of an exposure Report the incident to your PI immediately and to the Biosafety Officer Complete a Laboratory Incident and Accident Report Form (and, if appropriate, the Workman s Compensa>on form) These are in the back pocket of the Bisafety Manual and at fandm.edu/biosafety Occupational Injury / Illness Report
What is a BSL1 Lab? Primary Containment and Safety Equipment Biosafety Element Biosafety Level 1 Personal protec>ve equipment Gloves, lab coats and eye protec>on are used in BSL1 labs to protect workers Characteris>cs of the biohazardous material Planned manipula>ons Not known to consistently cause disease in healthy adults Standard Microbiological Prac>ces Primary containment and protecbon PPE: gloves, lab coat and eye protec>on are used X FaciliBes design (secondary containment) Handwashing sink, emergency shower and eyewash, autoclave 22
Proper Use of Gloves Gloves should NOT to be worn outside of the work area. DO NOT wear gloves to open doors or touch equipment (i.e. doorknobs, light switches and keyboards) that others will be handling without gloves. Gloves must be removed CORRECTLY prior to washing hands and leaving the laboratory. With both hands gloved, pinch the top of one glove, carefully pull It off so that it is inside out and hold it in the gloved hand. Using the ungloved hand, grab the inside top of the second glove and pull it off so that it is inside the first glove The one glove policy may be used when you must transport biohazardous materials outside of the lab 23
Proper Use of Gloves Gloves must be replaced as soon as they become contaminated, torn, punctured or compromised. Be extremely careful when using a flame - do not catch the gloves on fire! Disposable gloves cannot be reused. PPE must be disposed of as biohazardous waste. 24
Proper Use of Lab Coats and Goggles Lab coats should be BUTTONED to protect your street clothing You must NOT take poten>ally contaminated lab coats home. Handle soiled lab coats as licle as possible, using gloves. Place soiled lab coats in autoclavable bags; they may be laundered and reused a_er steriliza>on Eye/face protec>on should be used if splashes or sprays are a possibility. 25
It is important to minimize the producbon of splashes and aerosols! It has been es>mated that approximately 65% of laboratory acquired infec>ons are caused by aerosols of pathogenic microorganisms. What are Aerosols? Aerosols are solid or liquid par>cles suspended in the air (1 to 100 μm) Larger par>cles secle more rapidly becoming a risk for surface contact. Smaller par>cles can remain airborne for a long period of >me and spread wide distances. Smaller par>culates (1 to 10 μm) are also more easily inhaled.
Do I Ever Generate Aerosols? Absolutely! Here are some common laboratory procedures that may produce aerosols: blowing out pipettes dropping / breakage of culture containers carelessly removing protective gloves harvesting infected material flaming inoculating needles, slides or loops inserting a hot loop into a culture opening lyophilized cultures, culture plates, tubes and bottles pouring liquids removing stoppers stirring liquids streaking inoculum In addition, there are many devices that, if used incorrectly, may create aerosols, including: blenders and vortexers bottles and flasks centrifuges homogenizers needles and syringes pipettes pressurized vessels rubber stoppers shakers sonicators vacuum and aspirating equipment
It is important to minimize the producbon of splashes and aerosols! The use of sealed centrifuge rotors and tubes and biosafety cabinets may be recommended at BSL1 when aerosol genera>on is possible.
What is a BSL1 Lab? How should the lab be designed or organized? Secondary Containment A hand washing sink with soap, safety shower and eyewash sta>on must be available Biosafety Element Biosafety Level 1 Characteris>cs of the biohazardous material Not known to consistently cause disease in healthy adults Planned manipula>ons Standard Microbiological Prac>ces Primary containment and protecbon PPE: gloves, lab coat and eye protec>on are used FaciliBes design (secondary containment) Handwashing sink, emergency shower and eyewash, autoclave 29
Biosafety Levels 2, 3 And 4 Build On The PracBces And Containment Required in BSL1 Labs There are no BSL3 or BSL4 labs at F&M! High Hazard BSL4 BSL3 Low Hazard BSL2 BSL1 For comparison, what do BSL2/3/4 labs look like?
What is a Biological Safety Level 2 Lab? BSL2 is required when using: Human pathogens such as: Salmonella sp. Staphylococcus sp. Influenza virus Hepa>>s A virus Adenoviral and len>viral cloning vectors All human cell lines, >ssues and body fluids Biosafety Element Biosafety Level 1 Biosafety Level 2 CharacterisBcs of the biohazardous material Planned manipula>ons Primary containment and protec>on Not known to consistently cause disease in healthy adults Standard Microbiological Prac>ces PPE: gloves, lab coat and eye protec>on are used Associated with human disease which is rarely serious and for which preven>ve or therapeu>c interven>ons are o_en available. Human cell lines, body fluids BSL- 1 prac>ces plus Biosafety manual defining: Restricted access, Biohazard warning signs, Sharps precau>ons, Biowaste prac>ces, Medical surveillance & Spill Clean- up. Training BSL- 1 protec>on plus: Physical containment for splashes/ aerosoliza>on; Biosafety Cabinets: aka >ssue culture hoods Facili>es design (secondary containment) Handwashing sink, emergency shower and eyewash, autoclave Same as BSL1 31
What is a Biological Safety Level 3 Lab? Biosafety Element Biosafety Level 3 Characteris>cs of the biohazardous material Planned manipula>ons Associated with human disease which may have serious or lethal consequences BSL- 2 prac>ces plus: more restricted access; decontamina>on of waste and lab clothing, medical surveillance. Mycobacterium tuberculosis - TB Francisella tularensis - tularemia Rickettsia Rocky Mt spotted fever recombinant influenza virus high pathogenicity avian influenza) Primary containment and protec>on Facili>es design (secondary containment) BSL- 2 protec>on plus: Physical containment for all work; Biosafety Cabinets. Respirators may be required. Access through anteroom, self closing double doors, negabve air flow into labs, exhausted air is not recirculated.
What is a Biological Safety Level 4 Lab? Biosafety Element Biosafety Level 4 Characteris>cs of the biohazardous material Associated with human disease which pose a high risk of life threatening disease and may be aerosol transmi]ed. Hendravirus Ebola virus Planned manipula>ons BSL- 3 prac>ces plus: clothing change on entering and leaving Primary containment and protec>on Facili>es design (secondary containment) BSL- 3 protec>on plus: Physical containment for all work with full bodies, air supplied posibve pressure suit BSL3 B plus: separate building or zone, dedicated air supply and exhaust..
If you have any quesbons. ever Ask your lab professor/research advisor Check out the Biosafety Manual Ask the Biosafety Officer Dr. Frielle LSP332E 358-4600 dfrielle@fandm.edu